title: Overexpression of NADH-dependent fumarate reductase improves D-xylose fermentation in recombinant Saccharomyces cerevisiae.
abstract: Deviation from optimal levels and ratios of redox cofactors NAD(H) and NADP(H) is common when microbes are metabolically engineered. The resulting redox imbalance often reduces the rate of substrate utilization as well as biomass and product formation. An example is the metabolism of D-xylose by recombinant Saccharomyces cerevisiae strains expressing xylose reductase and xylitol dehydrogenase encoding genes from Scheffersomyces stipitis. This pathway requires both NADPH and NAD(+). The effect of overexpressing the glycosomal NADH-dependent fumarate reductase (FRD) of Trypanosoma brucei in D-xylose-utilizing S. cerevisiae alone and together with an endogenous, cytosol directed NADH-kinase (POS5Δ17) was studied as one possible solution to overcome this imbalance. Expression of FRD and FRD + POS5Δ17 resulted in 60 and 23 % increase in ethanol yield, respectively, on D-xylose under anaerobic conditions. At the same time, xylitol yield decreased in the FRD strain suggesting an improvement in redox balance. We show that fumarate reductase of T. brucei can provide an important source of NAD(+) in yeast under anaerobic conditions, and can be useful for metabolic engineering strategies where the redox cofactors need to be balanced. The effects of FRD and NADH-kinase on aerobic and anaerobic D-xylose and D-glucose metabolism are discussed.
authors.affiliation: VTT, Technical Research Centre of Finland, PO Box 1000, 02044, VTT, Finland, laura.salusjarvi@vtt.fi.
authors.forename: Laura
authors.initials: L
authors.lastname: Salusjärvi
authors.forename: Sanna
authors.initials: S
authors.lastname: Kaunisto
authors.forename: Sami
authors.initials: S
authors.lastname: Holmström
authors.forename: Maija-Leena
authors.initials: ML
authors.lastname: Vehkomäki
authors.forename: Kari
authors.initials: K
authors.lastname: Koivuranta
authors.forename: Juha-Pekka
authors.initials: JP
authors.lastname: Pitkänen
authors.forename: Laura
authors.initials: L
authors.lastname: Ruohonen
mesh_terms: D005285:Fermentation; D005947:Glucose / Q000378:metabolism; D060847:Metabolic Engineering; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009243:NAD / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics; D014993:Xylitol / Q000378:metabolism; D014994:Xylose / Q000378:metabolism*
chemical_list: D024101:Mitochondrial Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D009243:NAD; D014994:Xylose; D044925:Oxidoreductases Acting on CH-CH Group Donors; C020155:fumarate reductase (NADH); D017853:Phosphotransferases (Alcohol Group Acceptor); C060670:POS5 protein, S cerevisiae; D005947:Glucose; D010100:Oxygen; D014993:Xylitol